Organization of the Membrane Domain of the Human Liver Sodium/Bile Acid Cotransporter

Abstract

Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

“…If cholestasis persists, the liver will be damaged and serum bile salts will raise followed by an increase of plasma liver enzymes as a consequence of liver injury. In order to lower or prevent the toxicity of the retained bile salts, The structure of Ntcp has not yet been resolved, but experimental evidence derived from translation/insertion scanning, alanine insertion scanning and mutation of putative N-linked glycosylation site provides evidence for nine transmembrane spanning domains [108], which was later refined to seven transmembrane spanning domains and helices 7 and 8 forming an extracellular loop at the plasma membrane. This loop is essential for function [207].…”

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships

“…Membrane insertion and sorting of NTCP and ASBT Based on bioinformatic predictions and experimental data, it has been shown that NTCP and ASBT have an extracellular N-terminus, an odd number of transmembrane helices (seven or nine) and a cytoplasmic Cterminus (Hagenbuch et al 1991;Hagenbuch and Meier 1994;Stieger et al 1994;Dawson and Oelkers 1995;Hallén et al 1999;Hallén et al 2002b;Zhang et al 2004). Several potential N-glycosylation sites are present in NTCP, ASBT and SOAT proteins, and site-directed mutagenesis has revealed that only N 5 and N 11 in rat Ntcp (Hagenbuch 1997), and N 10 in human ASBT (Zhang et al 2004) are glycosylated.…”

“…Using N-glycosylation-scanning mutagenesis, Zhang and coworkers strongly suggested a model with seven transmembrane domains for human ASBT (Zhang et al 2004). In contrast, a nine-transmembrane domain arrangement was found by Hallén and coworkers for human ASBT and NTCP (Hallén et al 1999(Hallén et al , 2000(Hallén et al , 2002b. However, it remains unclear whether all of these nine transmembrane segments cross the membrane or whether two of them fold as lipidburied re-entrance loops in the plasma membrane (Hallén et al 2002b;Zahner et al 2003).…”

“…Such a mechanism would allow the introduction of charged molecules into the inner part of the cell membrane, from where they can be released into the intracellular compartment (Zahner et al 2003). This P-loop-based transport mechanism could also resolve the conflicting results from ASBT/NTCP topology predictions regarding the true number of transmembrane domains, i.e., two P-loop structures could fold as lipid-buried re-entrant loops in the plasma membrane but would not cross the membrane like a transmembrane domain (Hallén et al 2002b). According to this transport mechanism, Na + /bile acid cotransport by NTCP and ASBT would be the result of a series of ligand-induced conformational changes in the carrier proteins.…”

Mutational Analysis of Uncharged Polar Residues and Proline in the Distal One-Third (Thr130–Pro142) of the Highly Conserved Region of Mouse Slc10a2

“…A variation on the nine-TM model, in which the seventh and eighth helices are not membranespanning and associate with the outer surface of the plasma membrane, has also been proposed. [18][19][20][21][22][23] In either case, the residues submitted to mutational analysis in this study were mapped in the N-terminal half of a TM segment, viz., the extracellular half of TM3 and the cytosolic half of TM4 in the seven-TM or nine-TM model respectively. Helical wheel projection illustrates y To whom correspondence should be addressed.…”